Interplay between extra cellular signaling pathways and the DNA methylation patterns of repetitive DNA sequences

Abstract

A large part of the human genome comprises repetitive sequences, one of which are the α satellite repeats. These sequences are primarily located within the centromeric and pericentromeric regions and exhibit repressive epigenetic modification, such as DNA methylation. The elevated DNA methylation levels at α satellite repeats play a pivotal role in repressing the transcription of repetitive DNA sequences and facilitating the formation of heterochromatin structures. DNA methylation which leads to the repression of gene transcription and is essential for the maintenance of genomic and chromosomal stability is among the most extensively studied epigenetic modifications. The loss of DNA methylation at α satellites has been linked to numerous diseases including cancer. Despite the highly methylated nature of α satellites, active transcription is, under certain conditions, a characteristic of numerous satellite DNAs, including α-satellite DNA, giving rise to non-coding RNAs (ncRNA). At low levels, these ncRNAs are important for the proper functioning of the cells and the maintenance of the centromere and alterations of the ncRNA level are also associated with various diseases. However, despite the significance of the DNA methylation of α satellites, it largely remains unclear, which cellular signals can influence the DNA methylation status at these sites. One contributing factor to this uncertainty is the lack of appropriate methods to investigate the locus-specific methylation pattern at α satellites in living cells, due to methodological difficulties associated with analyzing these highly repetitive sequences. In this work, the previously designed biosensor from Lungu et al. (2017) based on fluorescence complementation was utilized to investigate the DNA methylation status of α satellite repeats in living cells. The sensor comprises a highly specific CRISPR/dCas9-based anchor domain that is designed to recognize the α-satellite repeats located on chromosome 9. Additionally, it includes a reader domain that recognizes DNA methylation. This sensor enables the analysis of locus-specific DNA methylation at single-cell level in living cells. By establishing stable expression of this sensor in the untransformed breast epithelial cell line MCF10A, a link between α satellite DNA methylation, its transcriptional status and cell density was identified. At high cell density, the cells exhibit a reduction in DNA methylation and an increase in ncRNA transcription. The results obtained from the sensor-based experiments were validated through methylation-sensitive restriction digest coupled with qPCR (MSRE-qPCR). During perturbation experiments, the pivotal role of the cellular adhesion protein E-cadherin as a key mediator of this signaling pathway was identified. When E-cadherin was functionally inactivated at high cell density, these cells, like at low cell density, exhibited increased DNA methylation. Furthermore, the involvement of the actin cytoskeleton was demonstrated, as the disruption of actin polymerization at high cell density likewise results in increased DNA methylation. Notably, the adaptation of DNA methylation in response to cell density changes was observed in both untransformed breast epithelial and breast cancer cells, both of which exhibit E-cadherin positive cell-cell-contacts, indicating a conserved mechanism. Interestingly, breast cancer cell lines lacking E-cadherin expression and contact inhibition, did not react on cell density changes by adapting their methylation signature at repeats. In this work a novel signaling pathway is unveiled that establishes a direct link between the integrity of epithelial cell-cell-contacts and the chromatin landscape. This new pathway could play a significant role in normal epithelial homeostasis and the findings presented in this work enhance the current understanding of homeostasis. Moreover, deregulation of the pathway could have implications for the onset and progression of cancer, which is characterized by a loss of cell-cell contacts, lack of contact inhibition and high epigenetic instability. Therefore, any disruptions in this pathway could potentially play a pivotal role in the progression of cancer.